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. 2015 Dec 3;6:10091. doi: 10.1038/ncomms10091

Figure 1. Measuring target engagement at intracellular HDACs.

Figure 1

(a) Illustration of intracellular target engagement assay. A permeable fluorescent tracer binds in dynamic equilibrium to an intracellular target protein fused to Nluc, resulting in BRET. Introduction of compounds that bind the same target cause the tracer to be displaced, resulting in a decrease in BRET. (b) The HDAC tracer was derived from the adduct of a broad-coverage HDAC inhibitor (SAHA, shown in black) and the NCT dye (shown in red). (c) Introduction of SAHA-NCT to cell medium resulted in specific and concentration-dependent intracellular BRET with Nluc fusion of various HDACs (classes I and IIb) expressed in HeLa cells, as measured using a microplate luminometer. A control HDAC6 CD2 construct encoding a binding-deficient mutant (H610A/H611A) showed weakened engagement with the tracer. BRET values at each tracer concentration were background-corrected by parallel measurements made in the presence of an excess of unmodified SAHA (20 μM) (as described in Methods and Supplementary Fig. 1a). Apparent tracer affinities were estimated using equation (1) and reported in Supplementary Table 1. Data are mean±s.e.m. of three independent experiments. (d) Luminescence images of HeLa cells expressing HDAC6 or HDAC10 fused to Nluc, shown with false colouring to represent light emission from 420–500 nm (donor) or above 590 nm (acceptor). Images were acquired in the presence of SAHA-NCT tracer alone (1 μM), or in combination with excess unlabelled SAHA (10 μM). Signal suppression caused by excess SAHA indicates specificity of the BRET signal. (e) Concentration-dependent attenuation of BRET from intracellular HDAC fusions with titration of SAHA in the presence of 1 μM SAHA-NCT tracer. Data were collected on a microplate luminometer and are mean±s.e.m. of three independent experiments. See Supplementary Table 1 and equations (2) and (3) for estimation of apparent affinities of SAHA to HDACs. (f) BRET measurements showing the relative affinity of HDAC inhibitors in HeLa cells (1 μM SAHA-NCT). See Supplementary Table 2 for relative compound affinities to individual HDACs. Data are mean±s.d. of four data points.